Our objectives for the next program period specific aims are, twofold. First, we will complete our high resolution 3D description of basic motile behavior, behavior in natural waves, and the behavioral responses to 1) a spatial gradient of cAMP, 2) simulated temporal waves of cAMP and 3) the rapid addition of 10-6 cAMP This description will include the 3D reconstructions and motion analysis of the cell surface, nucleus, pseudopods and uropods of cells, and dynamic 3D reconstruction and motion analysis of GFP-tagged F-actin, myosin II, myosin IA, myosin IB and SCAR, using 3D-DIAScon. In addition, we will expand this description to include two additional behaviors (filopodial override, and streaming) that follow single cell chemotaxis in the aggregation process. Then, using this arsenal of behavioral phenotypes, we will investigate through mutant analysis the roles of myosin heavy chain (MHC) dephosphorylation, myosin regulatory light chain (RLC) phosphorylation, specific MHC and RLC kinases, the myosin Is, SCAR, and a variety of additional regulatory and cytoskeletal elements in locomotion and chemotaxis. The results of these studies will then be incorporated into unique dynamic models of chemotaxis that identify independent pathways according to the phase of the wave from which they emerge and the specific behavioral response they elicit.